Patients with Rieger syndrome, present with dental hypoplasia associated with mutations in the novel Pitx2 homeobox transcription factor gene. Pitx2 provides a unique tool for studying the molecular control of tooth formation since it is selective expressed at the earliest stage of tooth development. However, the mechanisms that regulate Pitx2 expression and function are not known. The focus of this application is to understand the molecular/biochemical mechanisms of Pitx2 in tooth morphogenesis. Based on preliminary data, Pitx2 is a member of the bicoid-like homeobox gene family and has transcriptional transactivation activity. We propose to identify Pitx2 functional domains and test our hypothesis that the C-terminal 39 amino acids act as a DNA binding inhibitory region modulated by protein-protein interactions. We have shown that Pitx2 transcriptional activity is modulated by the pituitary specific POU homeodomain transcription factor, Pit-1. In tooth morphogenesis it is likely that additional factors are clearly needed for full Pitx2 transactivation activity, similar to Pit-1 in the developing pituitary. A major objective of this application will be to answer the question., what are the factors that regulate Pitx2 function? We propose to first establish a working model of Pitx2 functional domains. In this context we will ask how does the C-terminal region inhibit DNA binding and transactivation, and how do the naturally occurring Rieger mutations affect Pitx2 function? In the first set of experiments, we will ask if Pitx2 activity is controlled by physical interactions with oral epithelial transcription factors, as suggested by our preliminary studies using the Pit-1 protein. We will test interactions with known candidates that are co-expressed with Pitx2 in the dental epithelia, such as the Msx2 and Dlx2 homeodomain proteins. This approach will also enable us to identify novel interacting proteins. We will then test the hypothesis that these protein interactions are important for Pitx2 function. We will test Pitx2 activity using a minimal bicoid enhancer/TK promoter and with an apparent target gene, the Dlx2 promoter which contains multiple bicoid- like elements. Dlx2 is expressed in the developing tooth primordia after Pits2 and appears to be an excellent candidate for regulation by Pitx2. In the second set of experiments we will determine the effect of the Rieger syndrom mutations and phosphorylation on Pitx2 activity. These studies will provide the foundation for a better understanding of the molecular control of early tooth development.